Polymer-based sustained release device

ABSTRACT

This invention relates to compositions for the sustained release of biologically active polypeptides, and methods of forming and using said compositions, for the sustained release of biologically active polypeptides. The sustained release compositions of this invention comprise a biocompatible polymer having dispersed therein, a biologically active polypeptide and a sugar.

The invention claimed in this application was made by or on behalfAlkermes Controlled Therapeutics Inc. II and Amylin Pharmaceuticals,Inc., parties to a joint research agreement within the meaning of 35U.S.C. §103(c)(3) and 37 C.F.R. §1.104(c)(4)(ii).

BACKGROUND OF THE INVENTION

Numerous proteins and peptides, collectively referred to herein aspolypeptides, exhibit biological activity in vivo and are useful asmedicaments. Many illnesses or conditions require administration of asustained level of medicament to provide the most effective prophylacticand/or therapeutic effects. Sustained levels are often achieved by theadministration of biologically active polypeptides by frequentsubcutaneous injections, which often results in fluctuating levels ofmedicament and poor patient compliance.

As an alternative, the use of biodegradable materials, such as polymers,encapsulating the medicament can be employed as a sustained deliverysystem. The use of biodegradable polymers, for example, in the form ofmicroparticles or microcarriers, can provide a sustained release ofmedicament, by utilizing the inherent biodegradability of the polymer tocontrol the release of the medicament thereby providing a moreconsistent, sustained level of medicament and improved patientcompliance.

However, these sustained release devices can often exhibit high initialbursts of medicament and minimal release thereafter, resulting in serumdrug levels outside the therapeutic window and/or poor bioavailabilityof the medicament. In addition, the presence of polymer, physiologicaltemperatures and body response to the sustained release composition cancause the medicament to be altered (e.g., degraded, aggregated) therebyinterfering with the desired release profile for the medicament.

Further, methods used to form sustained release compositions can resultin loss of activity of the medicament due to the instability of themedicament and the degradative effects of the processing steps.Degradative effects are particularly problematic when the medicament isa polypeptide.

Therefore, a need exists for a means of administering biologicallyactive polypeptides in a sustained fashion wherein the amount ofpolypeptide delivered is at therapeutic levels, and retains activity andpotency for the desired period of release. While much work has beendeveloped that addresses these problems, novel solutions are required.

SUMMARY OF THE INVENTION

The invention relates to the discovery that superior release profiles(such as those characterized by a ratio of C_(max) to C_(ave) of about 3or less) can be achieved with a formulation containing few components byoptimizing the silicone oil to polymer ratio in the manufacturingprocess, thereby achieving a low pore volume. This invention relates tocompositions for the sustained release of agents, such as biologicallyactive polypeptides, and methods of forming and using such compositions,for the sustained release of biologically active polypeptides. Thesustained release compositions of this invention comprise abiocompatible polymer, an agent, such as a biologically activepolypeptide, and a sugar. The polypeptide and sugar are preferablydispersed in the polymer. The polypeptide and sugar can be dispersedseparately or, preferably, together. The sustained release compositionprovides a desirable and consistent release profile. In a particularembodiment, the profile is characterized as having a ratio of C_(max) toC_(ave) of about 3 or less. In a preferred embodiment, the biologicallyactive polypeptide is an antidiabetic or glucoregulatory polypeptide,such as GLP-1, GLP-2, exendin-3, exendin-4 or an analog, derivative oragonist thereof, preferably exendin-4. The sugar is preferably sucrose,mannitol or a combination thereof. A preferred combination includesexendin-4 and sucrose and/or mannitol.

Additionally or alternatively, the sustained release compositioncomprises a biocompatible polymer, an agent, such as a biologicallyactive polypeptide and a sugar wherein the composition has a total porevolume of about 0.1 mL/g or less. In a specific embodiment, the totalpore volume is determined using mercury intrusion porosimetry.

Additionally or alternatively, the sustained release compositionconsists essentially of or, alternatively consists of, a biocompatiblepolymer, exendin-4 at a concentration of about 3% w/w and sucrose at aconcentration of about 2% w/w. The biocompatible polymer is preferably apoly lactide coglycolide polymer.

The invention also includes a method for forming compositions for thesustained release of biologically active agents, such as polypeptides,which comprises forming a mixture by combining an aqueous phasecomprising water, an agent, such as a water soluble polypeptide, and asugar with an oil phase comprising a biocompatible polymer and a solventfor the polymer; forming a water-in-oil emulsion by, for example,sonicating or homogenizing, the mixture; adding silicone oil to themixture to form embryonic microparticles; transferring the embryonicmicroparticles to a quench solvent to harden the microparticles;collecting the hardened microparticles; and drying the hardenedmicroparticles. In a particular embodiment, the silicone oil is added inan amount sufficient to achieve a silicone oil to polymer solvent ratioof about 1.5:1. Additionally or alternatively, the polymer is present inthe oil phase at about 10% w/v or less.

The agent or polypeptide, e.g. exendin-4, can be present in thecomposition described herein at a concentration of about 0.01% to about10% w/w based on the total weight of the final composition. In addition,the sugar, e.g. sucrose, can be present in a concentration of about0.01% to about 5% w/w of the final weight of the composition.

The composition of this invention can be administered to a human, orother animal, by injection, implantation (e.g., subcutaneously,intramuscularly, intraperitoneally, intracranially, and intradermally),administration to mucosal membranes (e.g., intranasally, intravaginally,intrapulmonary or by means of a suppository), or in situ delivery (e.g.,by enema or aerosol spray).

When the sustained release composition has incorporated therein ahormone, particularly an anti-diabetic or glucoregulatory peptide, forexample, GLP-1, GLP-2, exendin-3, exendin-4 or agonists, analogs orderivatives thereof, the composition is administered in atherapeutically effective amount to treat a patient suffering fromdiabetes mellitus, impaired glucose tolerance (IGT), obesity,cardiovascular (CV) disorder or any other disorder that can be treatedby one of the above polypeptides or derivatives, analogs or agoniststhereof.

The use of a sugar in the sustained release compositions of theinvention improves the bioavailability of the incorporated biologicallyactive polypeptide, e.g, anti-diabetic or glucoregulatory peptides, andminimizes loss of activity due to instability and/or chemicalinteractions between the polypeptide and other components contained orused in formulating the sustained release composition, while maintainingan excellent release profile.

The advantages of the sustained release formulations as described hereininclude increased patient compliance and acceptance by eliminating theneed for repetitive administration, increased therapeutic benefit byeliminating fluctuations in active agent concentration in blood levelsby providing a desirable release profile, and a potential lowering ofthe total amount of biologically active polypeptide necessary to providea therapeutic benefit by reducing these fluctuations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the relationship between the average porediameter and the in vitro release for sustained release compositionsdescribed herein (A.S.=Ammonium Sulfate).

FIG. 2 is a graph showing the effect of porosity on the in vitro releaseof exendin-4 from microparticles and the impact that the processingconditions, namely the ratio of silicone oil to methylene chloride, hason the porosity of the microparticles formed.

FIGS. 3A-3B are scans of cryogenic SEMs for selected microparticleformulations described herein.

FIG. 4A-4D are scans of cryogenic SEMs for selected microparticleformulations described herein.

FIG. 5 is a plot of % residual ethanol and methylene chloride versus Tgfor microparticle formulations described herein.

FIG. 6 is a representative pharmacokinetic curve (concentration, pg/mlv. time, days with inset showing concentrations over first day) forFormulation 2-1 (3% exendin-4 and 2% sucrose), Formulation 1 (3%exendin-4 alone) and Formulation 4 (3% exendin-4 and 0.5% ammoniumsulfate).

FIG. 7 is a graph of in vivo release profile for the three microparticleFormulations 2, 2-1 and 2-2.

FIG. 8 is a graph of the pharmacokinetic data for microparticleFormulations 5-1, 5-2 and 5-3.

FIG. 9 is a graph illustrating the relationship between processparameters and the inner emulsion size achieved by the process.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to compositions for the sustained release ofbiologically active polypeptides, and methods of forming and using saidcompositions, for the sustained release of biologically activepolypeptides. The sustained release compositions of this inventioncomprise a biocompatible polymer, and agent, such as a biologicallyactive polypeptide, and a sugar. The agent and sugar are dispersed inthe biocompatible polymer separately or, preferably, together. In aparticular embodiment, the sustained release composition ischaracterized by a release profile having a ratio of maximum serumconcentration (C_(max)) to average serum concentration (C_(ave)) ofabout 3 or less. As used herein, the terms a or an refer to one or more.

The Agent

In a preferred embodiment, the agent is a biologically activepolypeptide such as an antidiabetic or glucoregulatory polypeptide,including GLP-1, GLP-2, exendin-3, exendin-4 or an analog, derivative oragonist thereof. Most specifically, the polypeptide is exendin-4.However, other agents can take advantage of the discoveries made herein.

Biologically active polypeptides as used herein collectively refers tobiologically active proteins and peptides and the pharmaceuticallyacceptable salts thereof, which are in their molecular, biologicallyactive form when released in vivo, thereby possessing the desiredtherapeutic, prophylactic and/or diagnostic properties in vivo.Typically, the polypeptide has a molecular weight between 500 and200,000 Daltons.

Suitable biologically active polypeptides include, but are not limitedto, glucagon, glucagon-like peptides such as, GLP-1, GLP-2 or other GLPanalogs, derivatives or agonists of Glucagon Like Peptides, exendinssuch as, exendin-3 and exendin-4, derivatives, agonists and analogsthereof, vasoactive intestinal peptide (VIP), immunoglobulins,antibodies, cytokines (e.g., lymphokines, monokines, chemokines),interleukins, macrophage activating factors, interferons,erythropoietin, nucleases, tumor necrosis factor, colony stimulatingfactors (e.g., G-CSF), insulin, enzymes (e.g., superoxide dismutase,plasminogen activator, etc.), tumor suppressors, blood proteins,hormones and hormone analogs and agonists (e.g., follicle stimulatinghormone, growth hormone, adrenocorticotropic hormone, and luteinizinghormone releasing hormone (LHRH)), vaccines (e.g., tumoral, bacterialand viral antigens), antigens, blood coagulation factors, growth factors(NGF and EGF), gastrin, GRH, antibacterial peptides such as defensin,enkephalins, bradykinins, calcitonin and muteins, analogs, truncation,deletion and substitution variants and pharmaceutically acceptable saltsof all the foregoing.

Exendin-4 is a 39 amino acid polypeptide. The amino acid sequence ofexendin-4 can be found in U.S. Pat. No. 5,424,286 issued to Eng on Jun.13, 1995, the entire content of which is hereby incorporated byreference. AC2993 and exenatide are synonymous with the term exendin-4.Exendin-4 has been shown in humans and animals to stimulate secretion ofinsulin in the presence of elevated blood glucose concentrations, butnot during periods of low blood glucose concentrations (hypoglycemia).It has also been shown to suppress glucagon secretion, slow gastricemptying and affect food intake and body weight, as well as otheractions. As such, exendin-4 and analogs and agonists thereof can beuseful in the treatment of diabetes mellitus, IGT, obesity, etc.

The amount of biologically active polypeptide, which is contained withinthe polymeric matrix of a sustained release composition, is atherapeutically, diagnostically or prophylactically effective amountwhich can be determined by a person of ordinary skill in the art, takinginto consideration factors such as body weight, condition to be treated,type of polymer used, and release rate from the polymer.

Sustained release compositions generally contain from about 0.01% (w/w)to about 50% (w/w) of the agent, e.g., biologically active polypeptide(such as exendin-4) (total weight of composition). For example, theamount of biologically active polypeptide (such as exendin-4) can befrom about 0.1% (w/w) to about 30% (w/w) of the total weight of thecomposition. The amount of polypeptide will vary depending upon thedesired effect, potency of the agent, the planned release levels, andthe time span over which the polypeptide will be released. Preferably,the range of loading is between about 0.1% (w/w) to about 10% (w/w), forexample, 0.5% (w/w) to about 5% (w/w). Superior release profiles wereobtained when the agent, e.g. exendin-4, was loaded at about 3% w/w.

The Sugar

A sugar, as defined herein, is a monosaccharide, disaccharide oroligosaccharide (from about 3 to about 10 monosaccharides) or aderivative thereof. For example, sugar alcohols of monosaccharides aresuitable derivatives included in the present definition of sugar. Assuch, the sugar alcohol mannitol, for example, which is derived from themonosaccharide mannose is included in the definition of sugar as usedherein.

Suitable monosaccharides include, but are not limited to, glucose,fructose and mannose. A disaccharide, as further defined herein, is acompound which upon hydrolysis yields two molecules of a monosaccharide.Suitable disaccharides include, but are not limited to, sucrose, lactoseand trehalose. Suitable oligosaccharides include, but are not limitedto, raffinose and acarbose.

The amount of sugar present in the sustained release composition canrange from about 0.01% (w/w) to about 50% (w/w), such as from about0.01% (w/w) to about 10% (w/w), such as from about 0.1% (w/w) to about5% (w/w) of the total weight of the sustained release composition.Excellent release profiles were obtained incorporating about 2% (w/w)sucrose.

Alternatively, the amount of sugar present in the sustained releasecomposition can be referred to on a weight ratio with the agent orbiologically active polypeptide. For example, the polypeptide and sugarcan be present in a ratio from about 10:1 to about 1:10 weight:weight.In a particularly preferred embodiment, the ratio of polypeptide (e.g.,exendin-4) to sugar (e.g., sucrose) is about 3:2 (w/w).

Combinations of two or more sugars can also be used. The amount ofsugar, when a combination is employed, is the same as the ranges recitedabove.

When the polypeptide is exendin-4, the sugar is preferably sucrose,mannitol or a combination thereof.

The Polymer

Polymers suitable to form the sustained release composition of thisinvention are biocompatible polymers which can be either biodegradableor non-biodegradable polymers or blends or copolymers thereof. A polymeris biocompatible if the polymer and any degradation products of thepolymer are non-toxic to the recipient and also possess no significantdeleterious or untoward effects on the recipient's body, such as asubstantial immunological reaction at the injection site.

Biodegradable, as defined herein, means the composition will degrade orerode in vivo to form smaller units or chemical species. Degradation canresult, for example, by enzymatic, chemical and physical processes.Suitable biocompatible, biodegradable polymers include, for example,poly(lactides), poly(glycolides), poly(lactide-co-glycolides),poly(lactic acid)s, poly(glycolic acid)s, polycarbonates,polyesteramides, polyanydrides, poly(amino acids), polyorthoesters,poly(dioxanone)s, poly(alkylene alkylate)s, copolymers or polyethyleneglycol and polyorthoester, biodegradable polyurethane, blends thereof,and copolymers thereof.

Suitable biocompatible, non-biodegradable polymers includenon-biodegradable polymers selected from the group consisting ofpolyacrylates, polymers of ethylene-vinyl acetates and other acylsubstituted cellulose acetates, non-degradable polyurethanes,polystyrenes, polyvinylchloride, polyvinyl flouride, poly(vinylimidazole), chlorosulphonate polyolefins, polyethylene oxide, blendsthereof, and copolymers thereof.

Acceptable molecular weights for polymers used in this invention can bedetermined by a person of ordinary skill in the art taking intoconsideration factors such as the desired polymer degradation rate,physical properties such as mechanical strength, end group chemistry andrate of dissolution of polymer in solvent. Typically, an acceptablerange of molecular weight is of about 2,000 Daltons to about 2,000,000Daltons. In a preferred embodiment, the polymer is biodegradable polymeror copolymer. In a more preferred embodiment, the polymer is apoly(lactide-co-glycolide) (hereinafter “PLG”) with a lactide:glycolideratio of about 1:1 and a molecular weight of about 10,000 Daltons toabout 90,000 Daltons. In an even more preferred embodiment, themolecular weight of the PLG used in the present invention has amolecular weight of about 30,000 Daltons to about 70,000 Daltons such asabout 50,000 to about 60,000 Daltons.

The PLGs can possess acid end groups or blocked end groups, such as canbe obtained by esterifying the acid. Excellent results were obtainedwith a PLG with an acid end group.

Polymers can also be selected based upon the polymer's inherentviscosity. Suitable inherent viscosities include about 0.06 to 1.0 dL/g,such as about 0.2 to 0.6 dL/g, more preferably between about 0.3 to 0.5dL/g. Preferred polymers are chosen that will degrade in 3 to 4 weeks.Suitable polymers can be purchased from Alkermes, Inc. under thetradename Medisorb®, such as those sold as 5050 DL 3A or 5050 DL 4A.Boehringer Ingelheim Resomer® PLGs may also be used, such as Resomer®RG503 and 503H.

The sustained release composition of this invention can be formed intomany shapes such as a film, a pellet, a cylinder, a disc or amicroparticle. A microparticle, as defined herein, comprises a polymercomponent having a diameter of less than about one millimeter and havingbiologically active polypeptide dispersed or dissolved therein. Amicroparticle can have a spherical, non-spherical or irregular shape.Typically, the microparticle will be of a size suitable for injection. Atypical size range for microparticles is 1000 microns or less. In aparticular embodiment, the microparticle ranges from about one to about180 microns in diameter.

Additional Excipients

While it is possible that additional excipients can be added to theformulations of the claimed invention as is well known in the art, asurprising discovery of the present invention is that an excellentrelease profile can be achieved with the simple formulation describedherein. Such additional excipients can increase or decrease the rate ofrelease of the agent. Ingredients which can substantially increase therate of release include pore forming agents and excipients whichfacilitate polymer degradation. For example, the rate of polymerhydrolysis is increased in non-neutral pH. Therefore, an acidic or abasic excipient such as an inorganic acid or inorganic base can be addedto the polymer solution, used to form the microparticles, to alter thepolymer erosion rate. Ingredients which can substantially decrease therate of release include excipients that decrease the water solubility ofthe agent.

A preferred embodiment of the described sustained release formulationsconsists essentially of the biocompatible polymer, the agent and thesugar. By “consists essentially of” is meant the absence of ingredientswhich substantially increase the rate of release of the active agentfrom the formulation. Examples of additional excipients which would notbe expected to substantially increase or decrease the rate of release ofthe agent include additional active agents and inert ingredients.

In yet another embodiment, the formulation consists of the biocompatiblepolymer, the agent and the sugar. By “consists of” is meant the absenceof components or ingredients other than those listed and residual levelsof starting materials, solvents, etc. from the process.

It has been a surprising discovery that buffering agents such asacetate, citrate, phosphate or other biologically compatible buffer wasnot necessary in the aqueous phase to achieve a sustained releaseformulation with agent, e.g., exendin-4, with good to excellentbioavailability. It was also a surprising discovery that salting outsalts were unnecessary to control burst of the agent, e.g., exendin-4.As such, the compositions of the invention also include compositions, asdescribed herein, in the substantial (or complete) absence of bufferand/or salting out salts.

Alternatively or additionally, the sustained release composition of theinvention has low porosity. In such embodiments, the sustained releasecomposition comprises a biocompatible polymer, a biologically activepolypeptide and a sugar wherein the composition has a total pore volumeof about 0.1 mL/g or less. In a specific embodiment, the total porevolume is determined using mercury intrusion porosimetry, e.g., asdescribed in more detail below.

Administration

The compositions of the invention can be administered according tomethods generally known in the art. The composition of this inventioncan be administered to a patient (e.g., a human in need of the agent) orother animal, by injection, implantation (e.g., subcutaneously,intramuscularly, intraperitoneally, intracranially, and intradermally),administration to mucosal membranes (e.g., intranasally, intravaginally,intrapulmonary or by means of a suppository), or in situ delivery (e.g.,by enema or aerosol spray).

The sustained release composition can be administered using any dosingschedule which achieves the desired therapeutic levels for the desiredperiod of time. For example, the sustained release composition can beadministered and the patient monitored until levels of the drug beingdelivered return to baseline. Following a return to baseline, thesustained release composition can be administered again. Alternatively,the subsequent administration of the sustained release composition canoccur prior to achieving baseline levels in the patient.

For example, when the sustained release composition has incorporatedtherein a hormone, particularly an anti-diabetic or glucoregulatorypeptide, for example, GLP-1, GLP-2, exendin-3, exendin-4 or agonists,analogs or derivatives thereof, the composition is administered in atherapeutically effective amount to treat a patient suffering fromdiabetes mellitus, IGT, obesity, cardiovascular (CV) disorder or anyother disorder that can be treated by one of the above polypeptides orderivatives, analogs or agonists thereof.

Other conditions which can be treated by administering the sustainedrelease composition of the invention include Type I and Type II diabeteswhich can be treated with a sustained release composition having insulinincorporated therein. In addition, when the incorporated polypeptide isFSH or analogs thereof the sustained release composition can be used totreat infertility. In other instances, the sustained release compositioncan be used to treat Multiple Sclerosis when the incorporatedpolypeptide is beta interferon or a mutein thereof. As can be realized,the sustained release composition can be used to treat disease whichresponds to administration of a given polypeptide.

In a further embodiment, the sustained release composition of thepresent invention can be coadministered with a corticosteroid.Coadministration of the sustained release composition of the inventionwith a corticosteroid can further increase the bioavailability of thebiologically active polypeptide of the sustained release composition.Coadministration of a corticosteroid in combination with sustainedrelease compositions is described in detail in U.S. Patent Application60/419,430 entitled, “Method of Modifying the Release Profile ofSustained Release Compositions” by Dasch et al. the entire content ofwhich is hereby incorporated by reference.

Corticosteroids, as defined herein, refers to steroidalanti-inflammatory agents also referred to as glucocorticoids.

Suitable corticosteroids include, but are not limited to,21-Acetoxypregnenolone, Alclometasone, Algestone, Amcinonide,Beclomethasone, Betamethasone, Budesonide, Chloroprednisone, Clobetasol,Clobetasone, Clocortolone, Cloprednol, Corticosterone, Cortisone,Cortivazol, Deflazacort, Desonide, Desoximetasone, Dexamethasone,Disflorasone, Diflucortolone, Difluprednate, Enoxolone, Fluazacort,Flucloronide, Flumethasone, Flunisolide, Flucinolone Acetonide,Fluocinonide, Fluocortin Butyl, Flucortolone, Fluorometholone,Fluperolone Acetate, Fluprednidene Acetate, Fluprednisolone,Flurandrenolide, Fluticasone Propionate, Formocortal, Halcinonide,Halobetasol Propionate, Halometasone, Halopredone Acetate,Hydrocortamate, Hydrocortisone, Loteprednol Etabonate, Mazipredone,Medrysone, Meprednisone, Methylprednisolone, Mometasone Furoate,Paramethasone, Prednicarbate, Prednisolone, Prednisolone25-Diethylamino-acetate, Prednisolone Sodium Phosphate, Prednisone,Prednival, Prednylidene, Rimexolone, Tixocortol, Triamcinolone (allforms), for example, Triamcinolone Acetonide, Triamcinolone Acetonide21-oic acid methyl ester, Triamcinolone Benetonide, TriamcinoloneHexacetonide, Triamcinolone Diacetate, pharmaceutically acceptablemixtures thereof and salts thereof and any other derivative and analogthereof.

In one embodiment, the corticosteroid can be co-incorporated into thesustained release composition comprising the biocompatible polymer andthe biologically active polypeptide agent incorporated therein.

In another embodiment, the corticosteroid can be separately incorporatedinto a second biocompatible polymer. The second biocompatible polymercan be the same or different from the first biocompatible polymer whichhas the biologically active polypeptide agent incorporated therein.

In yet another embodiment, the corticosteroid can be present in anunencapsulated state but commingled with the sustained releasecomposition. For example, the corticosteroid can be solubilized in thevehicle used to deliver the sustained release composition.Alternatively, the corticosteroid can be present as a solid suspended inan appropriate vehicle. Further, the corticosteroid can be present as apowder which is commingled with the sustained release composition.

It is understood that the corticosteroid is present in an amountsufficient to increase the bioavailability of the biologically activepolypeptide from the sustained release composition. Increasedbioavailability refers to an increase in the bioavailability of thebiologically active polypeptide from the sustained release compositionwhen co-administered with a corticosteroid in comparison to theadministration in the absence of corticosteroid over a time periodbeginning at two days post administration and ending at the end of therelease cycle for the particular formulation.

As used herein, patient refers to a human, such as a human in need ofthe agent or therapy, prophylaxis or diagnostic method.

As defined herein, a sustained release of biologically activepolypeptide is a release of the polypeptide from the sustained releasecomposition of the invention which occurs over a period which is longerthan that period during which a biologically significant amount of thepolypeptide would be available following direct administration of asolution of the polypeptide. It is preferred that a sustained release bea release which occurs over a period of at least about one week, such asat least about two weeks, at least about three weeks or at least aboutfour weeks. The sustained release can be a continuous or a discontinuousrelease, with relatively constant or varying rates of release. Thecontinuity of release and level of release can be affected by the typeof polymer composition used (e.g., monomer ratios, molecular weight,block composition, and varying combinations of polymers), polypeptideloading, and/or selection of excipients to produce the desired effect.

As used herein, a therapeutically effective amount, prophylacticallyeffective amount or diagnostically effective amount is the amount of thesustained release composition needed to elicit the desired biologicalresponse following administration.

C_(max) as used herein is the maximum serum concentration of drug whichoccurs during the period of release which is monitored.

C_(ave) as used herein, is the average serum concentration of drugderived by dividing the area under the curve (AUC) of the releaseprofile by the duration of the release.

It is preferred that the ratio of C_(max) to C_(ave) be about 3 or less.This profile is particularly desirable of anti-diabetic orglucoregulatory polypeptides, such as those described above. A ratio ofabout 3 or less can provide a C_(ave) in a therapeutic window whileavoiding adverse drug side effects which can result from higher ratios.

Bioavailability, as that term is used herein, refers to the amount oftherapeutic that reaches the circulation system. Bioavailability can bedefined as the calculated Area Under the Curve (AUC) for the releaseprofile of a particular polypeptide during the time period starting atpost administration and ending at a predetermined time point. As isunderstood in the art, the release profile is generated by graphing theserum levels of a biologically active agent in a subject (Y-axis) atpredetermined time points (X-axis). Bioavailability is often referred toin terms of % bioavailability, which is the bioavailability achieved fora particular polypeptide following administration of a sustained releasecomposition divided by the bioavailability achieved for a particularpolypeptide following intravenous administration of the same dose ofdrug, multiplied by 100.

A modification of the release profile can be confirmed by appropriatepharmacokinetic monitoring of the patient's serum for the presence ofthe biologically active polypeptide agent. For example, specificantibody-based testing (e.g., ELISA and IRMA), as is well known in theart, can be used to determine the concentration of certain biologicallyactive polypeptide agents in the patient's serum. An example of suchtesting is described herein for exendin-4.

Pharmacodynamic monitoring of the patient to monitor the therapeuticeffects of the agent upon the patient can be used to confirm retentionof the biological activity of the released agent. Methods of monitoringpharmacodynamic effects can be selected based upon the biologicallyactive polypeptide agent being administered using widely availabletechniques.

Manufacture

A number of methods are known by which sustained release compositions(polymer/biologically active polypeptide matrices) of the invention canbe formed, particularly compositions having low porosity as describedherein. Detailed procedures for some methods of microparticle formationare set forth in the Working Examples. In a preferred embodiment, themethod of the invention for forming a composition for the sustainedrelease of biologically active polypeptide includes forming a mixture bycombining an aqueous phase comprising water, agent, such as a watersoluble polypeptide, and a sugar with an oil phase comprising abiocompatible polymer and a solvent for the polymer; forming awater-in-oil emulsion; adding a coacervation agent, for example siliconeoil, vegetable oil or mineral oil to the mixture to form embryonicmicroparticles; transferring the embryonic microparticles to a quenchsolvent to harden the microparticles; collecting the hardenedmicroparticles; and drying the hardened microparticles. This process isgenerally referred to herein as a water-oil-oil process (W/O/O).

Preferably, the polymer can be present in the oil phase in aconcentration ranging from about 3% w/w to about 25% w/w, preferably,from about 4% w/w to about 15% w/w, such as from about 5% w/w to about10% w/w. Excellent results were obtained herein using a 6% w/wconcentration of PLG in the oil phase.

The polymer is generally combined with a polymer solvent. Where thepolymer is a PLG, such as those preferred herein, the polymer is addedto a solvent for PLG. Such solvents are well known in the art. Apreferred solvent is methylene chloride.

The agent and sugar are added in the aqueous phase, preferably in thesame aqueous phase. The concentration of agent is preferably 10 to 100mg/g, preferably between 50 to 100 mg/g. The concentration of sugar ispreferably 10 to 50 mg/g and 30 to 50 mg/g.

The two phases are then mixed to form an emulsion. It is preferred thatthe emulsion be formed such that the inner emulsion droplet size is lessthan about 1 micron, preferably less than about 0.7 microns, morepreferably less than about 0.5 microns, such as about 0.4 microns.Sonicators and homogenizers can be used to form such an emulsion.

A coacervation agent as used herein refers to any oil in which thepolymer solution (polymer and solvent) is not readily solubilized intoand thereby forms a distinct phase with the polymer solution. Suitablecoacervation agents for use in the present invention include, but arenot limited to, silicone oil, vegetable oil and mineral oil. In aparticular embodiment, the coacervation agent is silicone oil and isadded in an amount sufficient to achieve a silicone oil to polymersolvent ratio from about 0.75:1 to about 2:1. In a particularembodiment, the ratio of silicone oil to polymer is from about 1:1 toabout 1.5:1. In a preferred embodiment, the ratio of silicone oil topolymer is about 1.5:1.

The resulting mixture is added to a quench, which comprises a polymernon-solvent. Polymer non-solvents are generally well known in the art. Aparticularly preferred quench comprises a heptane/ethanol solventsystem.

Solid drug can also be encapsulated using a modified version of theprocess described above. This modified process can be referred to as asolid/oil/oil (S/O/O).

For example, solid exendin-4 was suspended in methylene chloridecontaining 6% PLG and sonicated for about four minutes on ice.Subsequent processing was conducted in a manner analogous to the W/O/Omethod.

The invention will now be further and specifically described by thefollowing examples.

EXEMPLIFICATIONS Microparticle Preparation I

The sustained release compositions described herein were prepared by aphase separation process. The general process is described below formicroparticles containing exendin-4 and sucrose for a 1 kg batch size.

A. Inner Water-in-Oil Emulsion Formation

A water-in-oil emulsion was created with the aid of a homogenizer.Suitable homogenizers include an in-line Megatron homogenizer MT-V 3-65F/FF/FF, Kinematica AG, Switzerland. The water phase of the emulsion wasprepared by dissolving exendin-4 and excipients such as sucrose inwater. The concentration of drug in the resulting solution can be fromabout 50 mg/g to about 100 mg/g. For example, when the drug isexendin-4, the concentration of drug in solution can be from about 30 gto about 60 g per 600 g of water. In a particular embodiment, 50 gexendin-4 and 20 g sucrose were dissolved in 600 g water for irrigation(WFI). The specified amounts listed above represent a nominal loadwithout adjustment to compensate for peptide content strength specificto the lot of exendin-4 used. The oil phase of the emulsion was preparedby dissolving PLGA polymer (e.g., 930 g of purified 50:50 DL4A PLGA(Alkermes, Inc.) in methylene chloride (14.6 kg or 6% w/w).

The water phase was then added to the oil phase to form a coarseemulsion with an overhead mixer for about three minutes. Then, thecoarse emulsion was homogenized at approximately 10,000 rpm at ambienttemperature. This resulted in an inner emulsion droplet size of lessthan 1 micron. It is understood that inner emulsion formation can beachieved using any suitable means. Suitable means of emulsion formationinclude, but are not limited to, homogenization as described above andsonication.

B. Coacervate Formation

A coacervation step was then performed by adding silicone oil (21.8 kgof Dimethicone, NF, 350 cs) over about a five minute time period to theinner emulsion. This is equivalent to a ratio of 1.5:1, silicone oil tomethylene chloride. The methylene chloride from the polymer solutionpartitions into the silicone oil and begins to precipitate the polymeraround the water phase containing exendin-4, leading tomicroencapsulation. The embryonic microspheres thus formed are soft andrequire hardening. Frequently, the embryonic microspheres are permittedto stand for a short period of time, for example, from about 1 minute toabout 5 minutes prior to proceeding to the microsphere hardening step.

C. Microsphere Hardening and Rinse

The embryonic microspheres were then immediately transferred into aheptane/ethanol solvent mixture. The volume of heptane/ethanol mixtureneeded can be determined based on the microsphere batch size, typicallya 16:1 ratio of methylene chloride to heptane/ethanol solvent. In thepresent example, about 210 kg heptane and 23 kg ethanol in a 3° C.cooled, stirred tank were used. This solvent mixture hardened themicrospheres by extracting additional methylene chloride from themicrospheres. This hardening step can also be referred to as quenching.After being quenched for 1 hour at 3° C., the solvent mixture is eitherdecanted and fresh heptane (13 Kg) is added at 3° C. and held for 1 hourto rinse off residual silicone oil, ethanol and methylene chloride onthe microsphere surface or pumped directly to the collection step.

D. Microsphere Drying and Collection

At the end of the quench or decant/wash step, the microspheres weretransferred and collected on a 12″ Sweco Pharmasep Filter/Dryer ModelPH12Y6. The filter/dryer uses a 20 micron multilayered collection screenand is connected to a motor that vibrates the screen during collectionand drying. A final rinse with heptane (6 Kg at 3° C.) was performed toensure maximum line transfer and to remove any excess silicone oil. Themicrospheres were then dried under vacuum with a constant purge ofnitrogen gas at a controlled rate according to the following schedule: 6hours at 3° C.; 6 hours ramping to 41° C.; and 84 hours at 41° C.

After the completion of drying, the microspheres were discharged into acollection vessel, sieved through a 150 μm sieve, and stored at about−20° C. until filling.

For all microparticle formulations which were prepared herein the amountof polypeptide, for example, exendin-4 and excipients present in theprepared formulations is expressed as a % (w/w) based on the finalweight of the sustained release composition. The % (w/w) is a nominalpercentage, except where indicated.

Microparticle Preparation II

A. Inner Water-in-Oil Emulsion Formation

A water-in-oil emulsion was created with the aid of a sonicator.Suitable sonicators include Vibracell VCX 750 with model CV33 probehead, Sonics and Materials Inc., Newtown, Conn. The water phase of theemulsion was prepared by dissolving exendin-4 and excipients such assucrose in water. The concentration of drug in the resulting solutioncan be from about 50 mg/ml to about 100 mg/ml. For example, when thedrug is exendin-4, the concentration of drug in solution can be fromabout 3.28 g to about 6.55 g per 65.5 g of water. In a particularembodiment, 5.46 g exendin-4 and 2.18 g sucrose were dissolved in 65.5 gwater for irrigation or WFI. The specified amounts listed aboverepresent a 4% overage to target load in order to compensate for lossesupon filter sterilization of the components. The oil phase of theemulsion was prepared by dissolving PLGA polymer (e.g., 97.7 g ofpurified 50:50 DL4A PLGA (Alkermes, Inc.)) in methylene chloride (1539 gor 6% w/v).

The water phase was then added to the oil phase over about a threeminute period while sonicating at 100% amplitude at ambient temperature.The water phase was pumped through a ¼″ stainless steel tube with a 1″HPLC tube end (ID=20/1000″) at 5 psig, added below the sonication probeinside the sonication zone. Reactor was then stirred at 1400 to 1600rpm, with additional sonication at 100% amplitude for 2 minutes,followed by a 30 second hold, and then 1 minute more of sonication. Thisresulted in an inner emulsion droplet size of less than 0.5 microns. Itis understood that inner emulsion formation can be achieved using anysuitable means. Suitable means of emulsion formation include, but arenot limited to, sonication as described above and homogenization.

B. Coacervate Formation

A coacervation step was then performed by adding silicone oil (2294 grof Dimethicone, NF, 350 cs) over about a three to five minute timeperiod to the inner emulsion. This is equivalent to a ratio of 1.5:1,silicone oil to methylene chloride. The methylene chloride from thepolymer solution partitions into the silicone oil and begins toprecipitate the polymer around the water phase containing exendin-4,leading to microencapsulation. The embryonic microspheres thus formedare soft and require hardening. Frequently, the embryonic microspheresare permitted to stand for a short period of time, for example, fromabout 1 minute to about 5 minutes prior to proceeding to the microspherehardening step.

C. Microsphere Hardening and Rinse

The embryonic microspheres were then immediately transferred into aheptane/ethanol solvent mixture. The volume of heptane/ethanol mixtureneeded can be determined based on the microsphere batch size. In thepresent example, about 22 kg heptane and 2448 g ethanol in a 3° C.cooled, stirred tank (350 to 450 rpm) were used. This solvent mixturehardened the microspheres by extracting additional methylene chloridefrom the microspheres. This hardening step can also be referred to asquenching. After being quenched for 1 hour at 3° C., the solvent mixturewas decanted and fresh heptane (13 Kg) was added at 3° C. and held for 1hour to rinse off residual silicone oil, ethanol and methylene chlorideon the microsphere surface.

D. Microsphere Drying and Collection

At the end of the rinse step, the microspheres were transferred andcollected on a 6″ diameter, 20 micron multilayered screen inside thecone shaped drying chamber which acted as a dead-end filter. A finalrinse with heptane (6 Kg at 4° C.) was performed to ensure maximum linetransfer. The microspheres were then dried with a constant purge ofnitrogen gas at a controlled rate according to the following schedule:18 hours at 3° C.; 24 hours at 25° C.; 6 hours at 35° C.; and 42 hoursat 38° C.

After the completion of drying, the microspheres are discharged into ateflon/stainless steel sterilized collection vessel attached to thedrying cone. The collection vessel is sealed, removed from the dryingcone and stored at −20±5° C. until filling. Material remaining in thecone upon disassembly for cleaning is taken for drug content analysis.The yield was approximately 100 grams of microspheres.

For all microparticle formulations which were prepared herein the amountof polypeptide, for example, exendin-4 and excipients present in theprepared formulations is expressed as a % (w/w) based on the finalweight of the sustained release composition. The % (w/w) is a nominalpercentage, except were indicated.

Polymer:

Examples of specific PLG polymers suitable for use are listed below. Allof the polymers employed in the following examples are set forth in thelist and all listed polymers were obtained from Alkermes, Inc. ofCincinnati, Ohio and can be described as follows:

-   -   Polymer 2A: Poly(lactide-co-glycolide); 50:50 lactide:glycolide        ratio; 12.3 kD Mol. Wt.; IV=0.15 (dL/g).    -   Polymer 4A: Poly(lactide-co-glycolide); 50:50 lactide:glycolide        ratio; Mol. Wt. 45-64 kD; IV=0.45-0.47 (dL/g).

PURIFICATION OF PLG: It is known in the art (See, for example, PeptideAcylation by Poly(a-Hydroxy Esters) by Lucke et al., PharmaceuticalResearch, Vol. 19, No. 2, p. 175-181, February 2002) that proteins andpeptides which are incorporated in PLG matrices can be undesirablyaltered (e.g., degraded or chemically modified) as a result ofinteraction with degradation products of the PLG or impurities remainingafter preparation of the polymer. As such, the PLG polymers used in thepreparation of the majority of microparticle formulations describedherein were purified prior to preparation of the sustained releasecompositions using art recognized purification methods.

Characterization Methods:

It has been determined that the following characterization methods aresuitable for identifying microparticles which will provide a desirablerelease profile of active agent.

SEM

SEM was used to assess the particle size, shape and surface features ofthe microparticles. SEM imaging was performed on a Personal SEM® system(ASPEX™, LLC). All samples were deposited via spatula on standard SEMstubs covered with carbon double-sided tape. Samples were sputter coatedwith Au for about 90 seconds at 18 mA emission current using a Model SC7620 “Mini” Sputter Coater (Energy Beam Sciences). All SEM imaging wasperformed utilizing a 20 KeV electron beam over a magnification range ofapproximately 250 to 2500×.

Cryogenic SEM

The cross-section of microparticles was studied using cryogenic SEM. Themicroparticle sample was mixed with HISTO PREP® Solution (Fischer) andkept in a cryostat at −20° C. overnight. The hardened microparticleswere mounted on a glass cover slip and then sectioned using a metalknife. The sectioned particles were mounted on aluminium stubs, sputtercoated with Platinum and Palladium and observed under a ScanningElectron Microscope (Phillips 525M). Visual observation of the sectionsprovides a method of determining the degree of porosity for themicroparticles.

Porosity Measurement-Mercury Intrusion

Pore volume distribution in microparticles was determined using a modelSutoPor IV 9500 Moden Mercury Intrusion Porosimeter (Micromeritics,Norcross, Ga.). Briefly, mercury was forced into a known amount ofmicroparticles in a penetrometer by applying pressure in a step-wisemanner up to a maximum pressure of 60,000 Psia. The volume of mercuryintruded into the pores at various pressures was measured. This methodquantifies the pore distribution in the microparticles. That is, thesize of the pores that are intruded is inversely related to the appliedpressure. The equilibrium of the internal and external forces on theliquid-solid-vapor system can be described by the Washburn equation. Therelationship between applied pressure and the pore size into whichmercury is forced to enter is described by:

$D = \frac{{- 4}\;\gamma\;\cos\;\theta}{P}$Where:

-   -   D=pore diameter    -   γ=surface tension (constant)    -   θ=contact angle (constant)    -   P=Pressure        Therefore, the size of the pore into which mercury will intrude        is inversely proportional to the applied pressure. Assuming that        all pores are tight cylinders, the average pore diameter        (D=4V/A) can be calculated by dividing pore volume (V=πD2h/4) by        the pore area (A=πDh).        Residual Solvents

A single method was used for quantitation of heptane, ethanol andmethylene chloride. The equipment consisted of an HP 5890 Series 2 gaschromatograph with an Rtx 1301, 30 cm×0.53 mm column. About 130 mgmicroparticles were dissolved in 10 ml N,N-dimethylformamide. Propylacetate was used as the internal standard. The sample preparation wasadjusted so that concentrations of methylene chloride as low as 0.03%can be quantitated.

Microparticle Preparation

The microparticle batches set forth in Table 1 were prepared asdescribed above at the 100 gram scale using the 4A polymer and a ratioof silicone oil to methylene chloride of either 1.5:1 or 1:1 and thesilicone oil had a viscosity of 350 cs. The amount of exendin-4 and theexcipients used in the formulation are also set forth in Table 1.

TABLE 1 In vitro Lot # Formulation burst (%) Remarks 02-019-147(#1) 0%Sucrose, 0% AS 0.40 1.5:1 Si Oil:MeCl₂ 02-019-167(#2) 2% Sucrose (F16)0.40 1.5:1 Si Oil:MeCl₂ 02-019-160(#2-1) 2% Sucrose (F16) 0.44 1.5:1 SiOil:MeCl₂ 02-019-164(#2-2) 2% Sucrose (F16) 0.45 1.5:1 Si Oil:MeCl₂02-030-08(#2-3) 2% Sucrose (F16) 0.80 1:1 Si Oil:MeCl₂ 02-030-01(#2-4)2% Sucrose (F16) 1.0 1:1 Si Oil:MeCl₂ 02-030-04(#2-5) 2% Sucrose (F16)1.1 1:1 Si Oil:MeCl₂ 02-019-136(#3-1) 2% Sucrose, 0.5% AS (F14) 1.350:50 Quench 02-019-115(#3-2) 2% Sucrose, 0.5% AS (F14) 2.2 1.5:1 SiOil:MeCl₂ 02-019-170(#4) 0% Sucrose, 0.5% AS 3.8 1.5:1 Si Oil:MeCl₂02-019-133A(#3-3) 2% Sucrose, 0.5% AS (F14) 12.7 100% Heptane Quench02-019-185(#5) 2% sucrose (F17) 0.5 5% drug load, (5% drug load) 1.5:1Si Oil:MeCl₂ 02-019-64 (#3-4) 2% Sucrose, 0.5% AS (F14) 0.5 1.5:1 SiOil:MeCl₂ 02-019-10(#3-5) 2% Sucrose, 0.5% AS (F14) 1.30 1:1 SiOil:MeCl₂ 02-001-196(#3-6) 2% Sucrose, 0.5% AS (F14) 2.70 1:1 SiOil:MeCl₂ 02-019-24(#3-7) 2% Sucrose, 0.5% AS (F14) 6.70 1:1 SiOil:MeCl₂ *ALL FORMULATIONS HAD 3% DRUG LOAD WITH THE EXCEPTION OF #5POROSITY

The total intrusion volume obtained from the mercury intrusionporosimetry and the calculated average pore diameters are given in TABLE2. The relationship between the average pore diameter and the in vitrorelease is shown in FIG. 1

TABLE 2 Total Pore Volume In vitro Average Pore Lot # (mL/g) burst (%)Diameter (μm) 02-019-147(#1) 0.033 0.40 0.0068 02-019-167(#2) 0.035 0.400.0069 02-019-160(#2-1) 0.037 0.44 0.0070 02-019-164(#2-2) 0.035 0.450.0070 02-030-08(#2-3) 0.036 0.80 0.0070 02-030-01(#2-4) 0.038 1.00.0073 02-030-04(#2-5) 0.039 1.1 0.0074 02-019-136(#3-1) 0.041 1.30.0073 02-019-115(#3-2) 0.039 2.2 0.0078 02-019-170(#4) 0.067 3.8 0.012502-019-133A(#3-3) 0.513 12.7 0.0277 02-019-64 (#3-4) 0.030 0.5 0.006002-019-10(#3-5) 0.060 1.30 0.0090 02-001-196(#3-6) 0.060 2.70 0.010002-019-24(#3-7) 0.180 6.70 0.0170

FIG. 1 shows the effect of ammonium sulfate on the in vitro initialrelease. The data indicate that in vitro initial release is correlatedto the microparticle pore diameter. Formulations made with ammoniumsulfate showed varying levels of in vitro release and variable porosityunlike the formulations without ammonium sulfate which exhibitedconsistent porosity and release. During the manufacturing ofmicroparticles the presence of ammonium sulfate in the aqueous phase cansalt-out the drug substance during the preparation of theinner-emulsion. The differences in the micro-environment of theprecipitates can contribute to the differences in porosity and hence thevariation in the initial release. The effect was not observed informulations prepared without ammonium sulfate. Formulations withsucrose and exendin-4 show a more desirable and consistent level ofinitial release as compared to formulations having exendin-4, sucroseand ammonium sulfate.

FIG. 2 further demonstrates the effect of porosity on the in vitrorelease and the impact that the processing conditions, namely the ratioof silicone oil to methylene chloride, has on the porosity of themicroparticles formed. Briefly, microparticle formulations preparedusing a silicone oil-to-methylene chloride ratio of 1:1 (Formulations2-4 and 2-5 of Table 1) have a higher initial release than the sameformulations prepared using a silicone-to-methylene chloride ratio of1.5:1 (Formulations 2, 2-1 and 2-2 of Table 1). FIG. 2 suggests that ahigher ratio of silicone oil-to-methylene chloride results in a lowerporosity which results in a lower initial release.

Cryogenic SEM

Cryogenic SEM analysis was conducted as described above on Formulationsof the Types 2, 3 and 5 of Table 1. FIGS. 3A-3B are scans of micrographsfor selected formulations of Type 2 (Formulation 2-2, FIG. 3A) and ofType 5 (5% exendin-4, 2% sucrose, FIG. 3B). FIGS. 4A-D are scans ofmicrographs for Formulations 3-4, 3-5, 3-6 and 3-7, respectively ofTable 1. Again the variation in porosity exhibited with the use ofammonium sulfate which can contribute to the variability in initialrelease, can be seen in the cryogenic SEM cross sections of FIGS. 4A-D.

Residual Solvent Levels

The level of residual solvents in a given formulation can impact the Tgof the formulation. Residual solvent levels were determined formicroparticle formulations of Types 2 and 5 of Table 1. A single methodwas used for quantitation of heptane, ethanol and methylene chloride.The equipment consisted of an HP 5890 Series 2 gas chromatograph with anRtx 1301, 30 m×0.53 mm column. About 130 mg microparticles weredissolved in 10 ml N,N-dimethylformamide. Propyl acetate was used as theinternal standard. The sample preparation was adjusted so thatconcentrations of methylene chloride as low as 0.03% can be quantitated.

FIG. 5 is a plot of % residual ethanol and methylene chloride forformulations of Types 2 and 5 of Table 1 (3 or 5% exendin-4, 2%sucrose). FIG. 5 shows that the Tg decreases as the amount of residualsolvent increases.

Preparation of Microparticles Having 3% Exendin-4 and 2% Sucrose

In view of the variation in porosity introduced by the presence ofammonium sulfate in the microparticle formulations and theidentification of porosity as a characteristic which significantlyimpacts initial release, ammonium sulfate was not pursued in furtherdiscovery.

Impact of Inner Emulsion Droplet Size

The following study was done to determine the impact of processparameters on forming the inner emulsion as well as stability of theresulting emulsion and resulting 24 hour in vitro release ofmicrospheres produced using the different process parameters. Inneremulsions of the water phase and solvent phase were formed by eithersonication as described above for the 100 gr scale or homogenizationusing an MT5000 homogenizer with a 36/4 generator (Kinematica AG,Switzerland) at either a low speed (10,800 rpm) or high speed (21,300rpm). Following inner emulsion formation by the different techniques,the emulsions were held in the reactor with gentle agitation with anoverhead stirrer for 5, 15 or 60 minutes prior to an aliquot beingremoved. Following the designated hold times, the inner emulsion wasfurther processed as described above into microparticles and then the 24hour in vitro release determined for each batch as described below.

Inner Emulsion Droplet Size Characterization can be Determined Using theHoriba Particle Size Analyzer

An aliquot of the inner emulsion was withdrawn from the reactor using aglass pipet. Using a transfer pipet, ˜30 drops of the inner emulsion wasadded to ˜10 ml of 6% Medisorb® 50:50 4A PLG polymer solution in a 20 ccscrew-cap scintillation vial followed by mixing. The 6% Medisorb® 50:504A PLG polymer solution also served as the reference blank solution.About 9 ml of this diluted emulsion sample was then transferred into aclean 10 ml Horiba sample holder. A cover was placed on the sampleholder to prevent rapid evaporation of the polymer solvent. The preparedsample was within the acceptable % transmission reading range of0.65%-0.90% per the blue bar (Lamp). A relative refractive index settingof 0.94-0.00i was selected in the program setup. The sample was thenmeasured by a Horiba particle size analyzer such as model LA 910 fordroplet size. The data correlating the process parameters and theachieved inner emulsion size over the 5, 15 and 60 minute hold times aswell as the resulting 24 hour in vitro release results (in parenthesis)are shown in FIG. 9.

Microsphere Characterization

Exendin-4 microspheres were routinely characterized with respect to drugcontent, particle size, residual solvents, initial in vitro release, andPK characteristics in rats. Drug was extracted to obtain a preliminaryassessment of exendin-4 purity post-encapsulation in selected batches.

In Vitro Initial Release

The initial release of exendin-4 was determined by measuring theconcentration of exendin-4 after 1 hour in release buffer (10 mM HEPES,100 mM NaCl, pH 7.4).

150±5 mg of microspheres were placed in 5.0 mL of 10 mM HEPES, 100 mMNaCl, pH 7.4 buffer at room temperature, vortexed for about 30 secondsto suspend the solution and then placed in a 37° C. air chamber for 1hour. After 1 hour, the samples were removed from the chamber andinverted several times to mix, followed by centrifuging at 3500 rpm for10 minutes. The supernatant was removed and analyzed immediately by HPLCusing the following conditions: Column: TSK-GEL®, 7.8 mm×30 cm, 5 m(TSOH BIOSEP PART #08540); Column Oven Temperature: Ambient; AutosamplerTemperature: 6° C.; Flow Rate: 0.8 mL/minute; Detection: 280 nm;Injection Volume: 10 L; Mobile Phase: 35% Acetonitrile/65% Water with0.1% TFA/liter (v/v); Run Time: Approximately 20 minutes. Exendin-4 bulkdrug substance, 0.2 mg/mL prepared in 30 mM Acetate Buffer, pH 4.5, wasused as a standard.Animal Studies

All pharmacokinetic (PK) studies described herein were conducted inadult male Sprague-Dawley rats weighing approximately 500±50 g.

For PK characterization of the microparticle formulations, each animalreceived a subcutaneous injection of microparticles suspended in diluent(3% carboxymethylcellulose, 0.9% NaCl, 0.1% Tween 20) to theinter-scapular region. Generally, the dose was approximately 1.0 mgexendin-4 per rat in an injection volume of 0.75 mL. Blood samples werecollected via lateral tail vein at 0.5, 2, 4, 6, 10, 24 hours, and 2, 4,7, 10, 14, 17, 21, 24 and 28 days post-dose. Blood samples wereimmediately placed in MICROTAINER® tubes containing EDTA and centrifugedat about 14,000×g for about two minutes. Plasma was then transferred toMICROTAINER® tubes without additive and stored at −70° C. until time ofassay. IRMA was used to determine plasma exendin concentrations.

In Vivo Release-IRMA

The method for quantifying exendin-4 in plasma is a sandwichimmunoassay, with the analyte captured by a solid phase monoclonalantibody EXE4:2-8.4 and detected by the radioiodinated monoclonalantibody GLP-1:3-3. Counts bound are quantitated from a standardcalibration curve. This assay is specific for full length or intactexendin-4 and does not detect exendin-4 (3-39). A typical standard curverange is 30 pg/mL to 2000 pg/mL depending on the age of the tracerantibody.

In Vitro and In Vivo Release

Formulations 2, 2-1 and 2-2 (3% exendin-4 and 2% sucrose) were testedfor initial release in vitro as described above. The in vitro releasewas 0.4%, 0.4% and 0.5%, respectively. All three batches also had arelatively low in vivo initial release in the range of 1154 to 1555pg/mL for C_(max) 0-1 day. FIG. 6 is a representative pharmacokineticcurve for the formulations having 3% exendin-4 and 2% sucrose (2-1) andalso for 3% exendin-4 alone (1) and 3% exendin-4 and 0.5% ammoniumsulfate (4). A ratio of silicone oil-to-methylene chloride of 1.5:1 wasused and the viscosity of the silicone oil was 350 cs.

From FIG. 6 it can be seen that the formulations not containing ammoniumsulfate exhibit a lower initial release. Although the formulation havingexendin-4 alone showed a suitable initial release the post encapsulationpurity of the drug was decreased as compared to the formulation havingthe exendin-4 in combination with the sucrose. The addition of sugar inthe formulations decreases degradation of the agent.

The in vivo release profile for the three formulations 2, 2-1 and 2-2compared above, are shown in FIG. 7. All three batches exhibited arelatively low initial release followed by a “trough” (low serum levelsbetween about day 4 to day 17), followed by a sustained release overabout day 21 to day 28. The low initial release and the shape of therelease profile were consistent for the three formulations.

Formulation Using a 1:1 Ratio of Silicone Oil to Methylene Chloride

Formulations 2-3, 2-4 and 2-5 from Table 1 (3% exendin-4, 2% sucrose)were prepared using a 1:1 ratio of silicone oil to methylene chloride.The initial release was higher for these formulations than forformulations 2, 2-1 and 2-2 of Table 1 (3% exendin-4, 2% sucrose with a1.5:1 silicone to methylene chloride ratio). Specifically the 1.5:1ratio formulations provided an average initial release about 0.4%,whereas the 1:1 ratio formulations provided an average initial releaseabout 1.0%. The same trend was observed in vivo with C_(max) 0-1 day inrats was 2288±520 pg/mL for a 1:1 ratio, whereas the C_(max) 0-1 day inrats was 1300±221 pg/mL for the 1.5:1 ratio.

Increased Drug Loading

Increasing the exendin-4 load to 4% while maintaining the sucrose at 2%resulted in an initial release in vitro and in vivo in the same range asfor the 3% loading.

Three formulations of Type 5 from Table 1 were prepared (5% drug load,2% sucrose, 1.5:1 silicone oil-to-methylene chloride ratio). The threebatches, 5-1, 5-2 and 5-3 all exhibited a low in vitro initial releaseranging from 0.2 to 0.5%. Similarly, the in vivo C_(max) of theformulations was consistently low ranging from 467 pg/mL to 1267 pg/mL.FIG. 8 shows a graph of the pharmacokinetic data for the three batchestested. Compared to the behavior of the 3% exendin-4 formulation having2% sucrose, the 5% formulations exhibited higher serum levels of drugover about day 1 and day 2. The remainder of the profile for the 5%formulations was similar to the 3% formulations having a trough followedby release of drug primarily over day 21 to day 28.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method of preparing a composition for thesustained release of a glucoregulatory peptide comprising the steps of:a) forming a mixture by combining: an aqueous phase comprising a sugarand a water-soluble glucoregulatory peptide, with an oil phasecomprising a biocompatible polymer and a solvent for the biocompatiblepolymer; b) forming a water-in-oil emulsion of the mixture from step a),wherein the inner emulsion droplet size is less than about 0.5 microns;c) adding a coacervation agent to the mixture to form embryonicmicroparticles, wherein the coacervation agent is silicone oil added inan amount sufficient to achieve a silicone oil to polymer solvent ratioof from about 0.75:1 to about 2:1; d) transferring the embryonicmicroparticles to a quench solvent to harden the microparticles; e)collecting the hardened microparticles; and f) drying the hardenedmicroparticles, wherein the glucoregulatory peptide is present in thecomposition at a concentration of about 0.01% to about 10% w/w based onthe total weight of the composition, and the sugar is present in thecomposition at a concentration of about 0.01% to about 5% w/w based onthe total weight of the composition.
 2. The method of claim 1, whereinwater-in-oil emulsion of step b) is formed by homogenization.
 3. Themethod of claim 1, wherein the inner emulsion droplet size is less thanor equal to about 0.4 microns.
 4. The method of claim 3, wherein theinner emulsion droplet size is from about 0.2 to about 0.4 microns. 5.The method of claim 1, wherein the glucoregulatory peptide is selectedfrom GLP-1, GLP-2, exendin-3, exendin-4, and combinations thereof. 6.The method of claim 1, wherein the sugar is selected from sucrose,mannitol, and combinations thereof.
 7. The method of claim 1, whereinthe biocompatible polymer is selected from the group consisting ofpoly(lactides), poly(glycolides), poly(lactide-co-glycolides),poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolicacid)s and blends and copolymers thereof.
 8. The method of claim 1,wherein the biocompatible polymer is present in the oil phase at about10% w/v or less.
 9. The method of claim 1, wherein in step c), thesilicone oil is added in an amount sufficient to achieve a silicone oilto polymer solvent ratio of from about 1:1 to about 1.5:1.
 10. Themethod of claim 9, wherein the ratio of silicone oil to polymer solventis about 0.5:1.
 11. The method of claim 1, wherein the solventbiocompatible polymer is methylene chloride.
 12. The method of claim 1,wherein the quench solvent is a heptane/ethanol solvent mixture.